This is the continuation of studies beginning 15 yrs ago that now include: identification of the first CD1 restricted T cells, identification of the first CD1 antigens as lipids and the first insights into CD1 trafficking. It has delineated the molecular, cell biological and functional aspects of this pathway of antigen presentation in humans. The current proposal is organized around defining Endogenous (infected cells) and Exogenous (antigens in extracellular fluid) pathways for lipid antigen presentation that determine the intracellular intersection of microbial lipid antigens with the CD1 isoforms that bind and present them to T cells. In Aim 1, we propose to determine the mechanism controlling the intemalization and trafficking of CD1a, the only CD1 isoform that lacks a tyrosine-based sorting motif but still follows a trafficking route of intemalization and return to the plasma membrane through recycling endosomes. Then, the intersection of CD1a presented lipid antigens (mycobactins) with CD1a in the recycling endosomes will be compared to the intersection of CD1b presented antigens (mycolates, LAM) with CD1b in the lysosomes. The trafficking of lipid antigens from live, virulent Mycobacterium tuberculosis or dead bacilli in phagosomes of DC will be examined to identify the antigen sampling compartments for CD1a and CD1b in infected cells (Aim 2). The trafficking of lipid antigens and CD1 isoforms in infected DC in which the bacteria are in phagosomes (Endogenous Lipid Antigen Presentation Pathway) will be compared in with the Exogenous Lipid Antigen Presentation Pathway (Aim 3) in which apolipoprotiens secreted by APC are shown to capture extracellular microbial lipid antigens and deliver them to the endocytic loading compartments of DC by specific receptor mediated uptake. Besides providing access to exogenous lipid antigens in general, this pathway enables uninfected bystander DC to acquire lipid antigens through the secretion and recapture mediated by apoE. These studies delineate the CD1 pathways of antigen presentation with relevance to microbial immunity and vaccine development.